Identification of PAK isoforms in platelets and inhibition of PAK4/5/6 phosphorylation but not the PAK1/2 phosphorylation by NSC23766. (A) Identification of PAK isoforms in platelets. Resting platelets were lysed and immunoblotted using specific anti-PAK1, anti-PAK2, anti-PAK3, and anti-PAK4 antibodies. Immunoblots showing PAK1, PAK2, PAK3, and PAK4 at their approximate molecular masses (*) in platelets. (B) Effect of 300 μM NSC23766 on PAK1/2 phosphorylation. Platelets stimulated with 0.5 U/mL thrombin in the absence (-■-) or in the presence of NSC23766 (-□-) were immunoblotted with anti–phospho-PAK1/2 antibody. Top, representative immunoblot of PAK1/2 phosphorylation (*). Bottom, graphic representation of PAK1/2 phosphorylation (between 55 and 75 kDa). Values are the mean + SD or mean − SD for 3 independent experiments. (C) Inhibition of class II-PAKs phosphorylation by 300 μM NSC23766. Platelets stimulated with thrombin in the absence (-■-) or in the presence of NSC23766 (-□-) were immunoblotted with anti–phospho-PAK4/5/6 antibody. Top, representative immunoblot of class II PAKs phosphorylation (*). Bottom, graphic representation of class II PAKs phosphorylation (between 75 and 100 kDa). Values are the mean + SD or mean − SD for 3 independent experiments. * denotes statistical significance (P < .001) with respect to nontreated sample.